Neuropeptides and their use for pest control

ABSTRACT

The present invention discloses novel pest control compounds comprising NPF polypeptides and methods for using such compounds in the control of pests.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.10/062,623, filed Jan. 31, 2002, which is a continuation-in-part of U.S.application Ser. No. 09/295,849, filed Apr. 21, 1999, each of which ishereby incorporated by reference herein in its entirety, including anyfigures, tables, nucleic acid sequences, amino acid sequences, anddrawings.

BACKGROUND OF THE INVENTION

Many blood-ingesting pests are known to feed on humans and animals, andmany pests are vectors for pathogenic microorganisms which threatenhuman and animal health, including commercially important livestock,pets and other animals. Various species of mosquitoes, for example,transmit diseases caused by viruses, and many are vectors fordisease-causing nematodes and protozoa. Mosquitoes of the genusAnopheles transmit Plasmodium, the protozoan which causes malaria, adevastating disease which results in approximately 1 million deathsannually. The mosquito species Aedes aegypti transmits an arbovirus thatcauses yellow fever in humans. Other arboviruses transmitted by Aedesspecies include the causative agents of dengue fever, eastern andwestern encephalitis, Venezuelan equine encephalitis, St. Louisencephalitis, chikungunya, oroponehe and bunyarnidera. The genus Culex,which includes the common house mosquito C. pipiens, is implicated inthe transmission of various forms of encephalitis and filarial worms.The common house mosquito also transmits Wuchereria bancrofti and Brugiamalayi, which cause various forms of lymphatic filariasis, includingelephantiasis. Trypanasoma cruzi, the causative agent of Chagas'disease, is transmitted by various species of blood-ingestingTriatominae bugs. The tsetse fly (Glossina spp.) transmits Africantrypanosomal diseases of humans and cattle. Many other diseases aretransmitted by various blood-ingesting pest species. The order Dipteracontains a large number of blood-ingesting and disease-bearing pests,including, for example, mosquitoes, black flies, no-see-ums (punkies),horse flies, deer flies and tsetse flies.

Various pesticides have been employed in efforts to control or eradicatepopulations of disease-bearing pests, such as disease-bearingblood-ingesting pests. For example, DDT, a chlorinated hydrocarbon, hasbeen used in attempts to eradicate malaria-bearing mosquitoes throughoutthe world. Other examples of chlorinated hydrocarbons are BHC, lindane,chlorobenzilate, methoxychlor, and the cyclodienes (e.g., aldrin,dieldrin, chlordane, heptachlor, and endrin). The long-term stability ofmany of these pesticides and their tendency to bioaccumulate render themparticularly dangerous to many non-pest organisms.

Another common class of pesticides is the organophosphates, which isperhaps the largest and most versatile class of pesticides.Organophosphates include, for example, parathion, MALATHION, diazinon,naled, methyl parathion, and dichlorvos. Organophosphates are generallymuch more toxic than the chlorinated hydrocarbons. Their pesticidaleffect results from their ability to inhibit the enzyme cholinesterase,an essential enzyme in the functioning of the insect nervous system.However, they also have toxic effects on many animals, including humans.

The carbamates, a relatively new group of pesticides, include suchcompounds as carbamyl, methomyl, and carbofuran. These compounds arerapidly detoxified and eliminated from animal tissues. Their toxicity isthought to involve a mechanism similar to the mechanism of theorganophosphates; consequently, they exhibit similar shortcomings,including animal toxicity.

A major problem in pest control results from the capability of manyspecies to develop pesticide resistance. Resistance results from theselection of naturally-occurring mutants possessing biochemical,physiological or behavioristic factors that enable the pests to toleratethe pesticide. Species of Anopheles mosquitoes, for example, have beenknown to develop resistance to DDT and dieldrin. DDT substitutes, suchas MALATHION, propoxur and fenitrothion are available; however, the costof these substitutes is much greater than the cost of DDT.

There is clearly a longstanding need in the art for pesticidal compoundsthat are pest-specific, that reduce or eliminate direct and/or indirectthreats to human health posed by presently available pesticides, thatare environmentally compatible in the sense that they are biodegradable,and are not toxic to non-pest organisms, and have reduced or no tendencyto bioaccummulate.

Many pests, including for example blood-inbibing pests, must consume anddigest a proteinaceous meal to acquire sufficient essential amino acidsfor growth, development and the production of mature eggs. Adult pests,such as adult mosquitoes, need these essential amino acids for theproduction of vitellogenins by the fat body. These vitellogenins areprecursors to yolk proteins which are critical components of oogenesis.Many pests, such as house flies and mosquitoes, produce oostatichormones that inhibit egg development by inhibiting digestion of theprotein meal, and thereby limiting the availability of the essentialamino acids necessary for egg development.

Serine esterases such as trypsin and trypsin-like enzymes (collectivelyreferred to herein as “TTLE”) are important components of the digestionof proteins by insects. In the mosquito, Aedes aegypti, an early trypsinthat is found in the midgut of newly emerged females is replaced,following the blood meal, by a late trypsin. A female mosquito typicallyweighs about 2 mg and produces 4 to 6 μg of trypsin within several hoursafter ingesting blood meal. Continuous biosynthesis at this rate wouldexhaust the available metabolic energy of a female mosquito; as aresult, the mosquito would be unable to produce mature eggs, or even tofind an oviposition site. To conserve metabolic energy, the mosquitoregulates TTLE biosynthesis with a peptide hormone named TrypsinModulating Oostatic Factor (TMOF). Mosquitoes produce TMOF in thefollicular epithelium of the ovary 12-35 hours after a blood meal; TMOFis then released into the hemolymph where it binds to a specificreceptor on the midgut epithelial cells, signaling the termination ofTTLE biosynthesis.

This regulatory mechanism is not unique for mosquitoes; flesh flies,fleas, sand flies, house flies, dog flies and other pests which ingestprotein as part of their diet have similar regulatory mechanisms.

In 1985, Borovsky purified an oostatic hormone 7,000-fold and disclosedthat injection of a hormone preparation into the body cavity of bloodimbibed mosquitoes caused inhibition of egg development and sterility(Borovsky, D. [1985] Arch. Insect Biochem. Physiol. 2:333-349).Following these observations, Borovsky (Borovsky, D. [1988] Arch. Ins.Biochem. Physiol. 7:187-210) reported that injection or passage of apeptide hormone preparation into mosquitoes inhibited the TTLEbiosynthesis in the epithelial cells of the gut. This inhibition causedinefficient digestion of the blood meal and a reduction in theavailability of essential amino acids translocated by the hemolymph,resulting in arrested egg development in the treated insect. Borovskyobserved that this inhibition of egg development does not occur when theoostatic hormone peptides are inside the lumen of the gut or other partsof the digestive system (Borovsky, D. [1988], supra).

Following the 1985 report, the isolated hormone, (a ten amino acidpeptide) and two TMOF analogues were disclosed in U.S. Pat. Nos.5,011,909 and 5,130,253, and in a 1990 publication (Borovsky, et al.[1990] FASEB J. 4:3015-3020). Additionally, U.S. Pat. No. 5,358,934discloses truncated forms of the full length TMOF which have prolinesremoved from the carboxy terminus, including the peptidesTyr-Asp-Pro-Ala-Pro (SEQ ID NO. 25), Tyr-Asp-Pro-Ala-Pro-Pro (SEQ ID NO.26), Tyr-Asp-Pro-Ala-Pro-Pro-Pro (SEQ ID NO. 27), andTyr-Asp-Pro-Ala-Pro-Pro-Pro-Pro (SEQ ID NO. 28).

Neuropeptides Y (NPY) are an abundant family of peptides that are widelydistributed in the central nervous system of vertebrates. NPY peptideshave also been recently isolated and identified in a cestode, aturbellarian, and in terrestrial and marine molluscs (Maule et al., 1991“Neuropeptide F: A Novel Parasitic Flatworm Regulatory Peptide fromMoniezia expansa (Cestoda: Cyclophylidea)” Parasitology 102:309-316;Curry et al., 1992 “Neuropeptide F: Primary Structure from theTurbellarian, Arthioposthia triangulata” Comp. Biochem. Physiol.101C:269-274; Leung et al., 1992 “The Primary Structure of NeuropeptideF (NPF) from the Garden Snail, Helix aspersa” Regul. Pep. 41:71-81;Rajpara et al., 1992 “Identification and Molecular Cloning ofNeuropeptide Y Homolog that Produces Prolonged Inhibition in AplysiaNeurons” Neuron. 9:505-513).

Invertebrate NPYs are highly homologous to vertebrate NPYs. The majordifference between vertebrate and invertebrate NPYs occurs at theC-terminus where the vertebrate NPY has an amidated tyrosine (Y) whereasinvertebrates have an amidated phenylalanine (F). Because of thisdifference, the invertebrate peptides are referred to as NPF peptides.

Cytoimmunochemical analyses of NPY peptides suggest that they areconcentrated in the brain of various insects, including the Coloradopotato beetle Leptinotarsa decemlineata (Verhaert et al., 1985 “DistinctLocalization of FMRFamide- and Bovine Pancreatic Polypeptide-LikeMaterial in the Brain, Retrocerebal Complex and Subesophageal Ganglionof the Cockroach Periplaneta americana” L. Brain Res. 348:331-338;Veenstra et al., 1985 “Immunocytochemical Localization of PeptidergicNeurons and Neurosecretory Cells in the Neuro-Endocrine System of theColorado Potato Beetle with Antisera to Vertebrate Regulatory Peptides”Histochemistry 82:9-18). Partial purification of NPY peptides in insectssuggests that both NPY and NPF are synthesized in insects (Duve et al.,1981 “Isolation and Partial Characterization of PancreaticPolypeptide-like Material in the Brain of the Blowfly alliphoravomitoria” Biochem. J. 197, 767-770).

Researchers have recently isolated two neuropeptides with NPF-likeimmunoreactivity from brain extracts of the Colorado potato beetle. Theresearchers purified the peptides using C₁₈ reversed phase high-pressureliquid chromatography (HPLC), and determined their structure using massspectrometry. The deduced structures of these peptides are:Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 1) andAla-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 2) designated NPF Iand NPF II, respectively (Spittaels, Kurt, Peter Verhaert, Chris Shaw,Richard N. Johnston et al. [1996] Insect Biochem. Molec. Biol.26(4):375-382).

BRIEF SUMMARY OF THE INVENTION

The subject invention pertains to materials and methods for controllingpests. In a preferred embodiment, the subject invention involves the useof a polypeptide comprising an NPF peptide to control pests (referred toherein as the “NPF polypeptides”). Specifically exemplified are NPFpolypeptides comprising an amino acid sequence selected from the groupconsisting of Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-amide (SEQ IDNO. 1) and Ala-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 2) andcorresponding non-amidated NPF polypeptidesAla-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 3) andAla-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 4), compositions comprisingsuch NPF polypeptides and methods for using such compounds andpesticidal compositions. In a preferred mode, the NPF polypeptidescomprise an amino acid sequence which consists of a native NPF peptideor a fragment, analogue, derivative or other functional equivalent of anNPF peptide.

Further exemplified NPF polypeptides include those comprising an aminoacid sequence selected from the group consisting ofArg-Pro-Pro-Thr-Arg-Phe-Arg-Phe-amide (SEQ ID NO. 5),Arg-Pro-Pro-Thr-Arg-Phe-Arg-Phe (SEQ ID NO. 6),Ala-Pro-Gln-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 7),Ala-Pro-Gln-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 8),Ala-Asn-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 9),Ala-Asn-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 10),Ala-Asp-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 11),Ala-Asp-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 12),Pro-Ile-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 13),Pro-Ile-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 14),Ala-Gln-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 15),Ala-Gln-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 16),Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 17),Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 18),Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 19),Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 20), Leu-Arg-Leu-Arg-Phe-amide(SEQ ID NO. 21), Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 22), Arg-Pro-Pro-Thr(SEQ ID NO. 23), and Arg-Phe-Arg-Phe (SEQ ID NO. 24). NPF polypeptidesof SEQ ID NOs. 5, 7, 9, 11, 13, and 15 are native NPF peptides frommosquito, American cockroach, or fruitfly, and are homologous with theamino acid sequences of SEQ ID NOs. 1 and 2, which are native to theColorado potato beetle. NPF polypeptides of SEQ ID NOs. 6, 8, 10, 12,14, and 16 are the respective non-amidated versions of these native NPFpeptides. Specifically exemplified NPF analogues are those comprising anamino acid sequence selected from the group consisting of SEQ ID NOs.17-24.

The NPF polypeptides of the subject invention are particularly activeagainst blood-ingesting pests, e.g., species of mosquitoes such as Aedesaegypti, which are vectors of many arthropod-borne viral diseases(arboviruses). These pests utilize serine esterases, such as TTLE astheir primary blood digesting enzymes.

One aspect of the subject invention pertains to methods for controllingblood-ingesting pests by applying to a pest or to a pest-inhabitedlocus, a pesticidal formulation comprising (a) an NPF polypeptidecomprising a native NPF peptide or functional equivalent thereof and (b)a pesticidally effective carrier.

The subject invention further pertains to the use of NPF polypeptides tocontrol other pests, including non-blood ingesting agricultural pests.These pests include, for example, coleopterans (beetles), lepidopterans(caterpillars), and mites. The compounds of the subject invention canalso be used to control household pests including, but not limited to,ants and cockroaches.

The present invention also includes addition salts, complexes andprodrugs such as esters of the NPF polypeptides, especially the nontoxicpharmaceutically or agriculturally acceptable acid addition salts. Theacid addition salts can be prepared in standard manner in a suitablesolvent from the parent compound and an excess of an acid, such ashydrochloric, hydrobromic, sulfuric, phosphoric, acetic, maleic,succinic, ethanedisulfonic or methanesulfonic acids. Also, theN-terminus and C-terminus of the NPF polypeptides can be chemicallymodified to further inhibit proteolysis by metabolic enzymes, forexample, by N-terminal carboxylation and/or C-terminal amidation.

Dextrorotory amino acids can also be usefully employed in the NPFpolypeptides of the present invention to inhibit the ability ofproteases to degrade the peptides of the subject invention.

NPF polypeptides in which only conservative substitutions have been madeare also provided by the present invention. Analogues of the NPFpolypeptides which have one or more amino acid substitutions forming abranched peptide (e.g., by substitution with an amino acid or amino acidanalogue having a free amino- or carboy-side chain that forms a peptidebond with a sequence of one or more amino acids, including but notlimited to prolines) or allowing circularization of the peptide (e.g.,by substitution with a cysteine, or insertion of a cysteine at theamino- or carboxy-terminus or internally, to provide a sulfhydryl groupfor disulfide bond formation), are also provided.

Also, derivation of the NPF polypeptides with long chain hydrocarbonsfacilitates passage through the cuticle into the pest body cavity.Therefore, a further embodiment of the subject invention pertains tocompositions comprising the NPF polypeptides bound to lipids or othercarriers.

Analogues and derivatives, and other functional equivalents, includedwithin the scope of the invention are those which retain some or all ofthe pesticidal activity of native NPF peptides, or those which showimproved activity as compared to a corresponding native NPF peptide.Thus, included within the scope of the invention are pesticidally activenative NPF peptides, fragments, analogues (e.g., homologues),derivatives, or other functional equivalents of native NPF peptides.

Yet another aspect of the subject invention pertains to DNA sequencesencoding the peptides of the subject invention disclosed herein. TheseDNA sequences can be readily synthesized by a person skilled in the art.The sequences may be used to transform an appropriate host to conferupon that host the ability to express the NPF polypeptides. Hosts ofparticular interest include bacteria, algae, yeasts, insect viruses, andplants. For each of these hosts, the DNA sequences may be specificallydesigned by a person skilled in the art to utilize codons known to beoptimally expressed in the particular hosts. Advantageous promoters canalso easily be utilized. Bacteria, yeasts, plants, algae, viruses, andother hosts each may be used to produce peptide for further use, orthese hosts can be used as vehicles for direct application of thepeptide to the target pest. A plant species can be transformed toexpress the NPF polypeptides, resulting in a plant variety that is toxicto a target pest species which feeds on the plant. Pest control isachieved when the pest ingests the transformed plant material therebyexposing the pest to the NPF polypeptide. Methods for transforming plantcells utilizing, for example Agrobacteria, are well known to thoseskilled in the art.

Another aspect of the subject invention pertains to a method ofcontrolling pests comprising administering to said pest an effectiveamount of one or more NPF polypeptides.

The subject invention provides pest control compositions wherein the NPFpolypeptides are formulated for application to the target pests or theirsitus. In a specific embodiment, the present invention providesrecombinant hosts, which express an NPF polypeptide. The recombinanthost can be a procaryotic or eucaryotic cell, including for example,yeast or algae cells which are transformed to express an NPFpolypeptide. The transformed host may also be a virus. The transformedhost can be applied to a pest's habitat, (e.g., where the target pest isa mosquito, the transformed host can be applied to a body of water whichserves as a habitat for mosquito larvae), where the pest will ingest thetransformed host resulting in control of the pest by the NPFpolypeptide.

The methods and materials of the subject invention provide a novelapproach to controlling pests and pest-transmitted diseases.

As used herein, the term “pesticidally effective” is used to indicate anamount or concentration of a pesticide, e.g., an NPF polypeptide, whichis sufficient to reduce the number of pests in a geographical locus, ascompared to a corresponding geographical locus in the absence of theamount or concentration of the pesticide.

The term “pesticidal” is not intended to refer only to the ability tokill pests, but also includes the ability to interfere with a pest'slife cycle in any way that results in an overall reduction in the pestpopulation. For example, the term “pesticidal” includes inhibition orelimination of reproductive ability of a pest, as well as inhibition ofa pest from progressing from one form to a more mature form, e.g.,transition between various larval instars or transition from larvae topupa or pupa to adult. Further, the term “pesticidal” is intended torefer to all phases of a pest life cycle; thus, for example, the termincludes larvicidal, ovicidal and adulticidal action.

The word “transform” is broadly used herein to refer to introduction ofan exogenous polynucleotide sequence into a prokaryotic or eukaryoticcell by any means known in the art (including for example, directtransmission of a polynucleotide sequence from a cell or virus particle,transmission by infective virus particles, and transmission by any knownpolynucleotide-bearing substance) resulting in a permanent or temporaryalteration of genotype and in an immortal or non-immortal cell line.

The terms “peptide,” “polypeptide,” and “protein” as used herein areintended to refer to amino acid sequences of any length.

Without intending to be bound by theory, the current invention is basedon the determination that NPF adversely affects TTLE biosynthesis in themidgut of female Aedes aegypti fed a blood meal and injected with NPFpolypeptide. Because the structure of NPF is different from TMOF itappears that NPF does not bind to a TMOF-specific binding site on thegut receptor but to a different site on the same or different receptor.Furthermore, cytoimmunochemical analysis, by the inventors, of themosquito gut after the blood meal, using antiserum against NPF, hassurprisingly revealed exocrine cells with NPF-like molecules that aresynthesized by mosquito epithelial cells 24 hours after a blood meal.NPF therefore appears to be a secondary signal in a cascade of signals:first TMOF is released from the ovary, TMOF then binds to a TMOF gutreceptor (Borovsky et al. [1994] FASEB J. 8:350-355) that stimulates thesynthesis and release of NPF from gut specific exocrine cells. NPF thenbinds to a receptor site on the gut at a site which may be adjacent toor part of the TMOF receptor, resulting cessation of biosynthesis ofTTLE. This surprising discovery opens the door to a new generation ofNPF pesticides, which inhibit biosynthesis of TTLE in a more directmanner than previously disclosed TMOF peptides.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO. 1 is an amidated neuropeptide designated NPF I.

SEQ ID NO. 2 is an amidated neuropeptide designated NPF II.

SEQ ID NO. 3 is a non-amidated version of the NPF I peptide.

SEQ ID NO. 4 is a non-amidated version of the NPF II peptide.

SEQ ID NOs. 5-24 are amidated and non-amidated versions of NPFpolypeptides.

SEQ ID NOs. 25-28 are TMOF peptides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the inhibitory affect of NPF I on trypsinbiosynthesis when injected into whole mosquitos. NPF I resulted in a 50%inhibition of trypsin biosynthesis when injected at a 1×10⁻⁶Mconcentration.

FIG. 2 is a graph showing the inhibitory affect of NPF II on trypsinbiosynthesis when injected into whole mosquitos. NPF II resulted in a35% inhibition of trypsin biosynthesis at a 1×10⁻³ M concentration.

FIG. 3 is a graph showing the inhibitory affect of NPF I on trypsinbiosynthesis when injected into ligated mosquito abdomens. NPF Iresulted in a 54% inhibition of trypsin biosynthesis at a 1×10⁻⁶ Mconcentration. These results strongly suggest that NPF acts on areceptor in the gut and not through a cell signaling transductionpathway which originates in the brain.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention concerns NPF polypeptides that can be used tocontrol target pests. Specifically exemplified is the use of NPFpolypeptides in controlling mosquitos and other pests. A method ofcontrolling pests is also specifically exemplified herein, which methodemploys the use of NPF I and/or NPF II, which have the sequencesAla-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 1) andAla-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 2), respectively, aswell as non-amidated versions of NPF I and NPF II,Ala-Arg-Gly-Pro-Gln-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 3) andAla-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 4), respectively.

Further exemplified NPF polypeptides include those comprising an aminoacid sequence selected from the group consisting ofArg-Pro-Pro-Thr-Arg-Phe-Arg-Phe-amide (SEQ ID NO. 5),Arg-Pro-Pro-Thr-Arg-Phe-Arg-Phe (SEQ ID NO. 6),Ala-Pro-Gln-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 7),Ala-Pro-Gln-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 8),Ala-Asn-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 9),Ala-Asn-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 10),Ala-Asp-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 11),Ala-Asp-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 12),Pro-Ile-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 13),Pro-Ile-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 14),Ala-Gln-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 15),Ala-Gln-Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 16),Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 17),Arg-Ser-Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 18),Pro-Ser-Leu-Arg-Leu-Arg-Phe-amide (SEQ ID NO. 19),Pro-Ser-Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 20), Leu-Arg-Leu-Arg-Phe-amide(SEQ ID NO. 21), Leu-Arg-Leu-Arg-Phe (SEQ ID NO. 22), Arg-Pro-Pro-Thr(SEQ ID NO. 23), and Arg-Phe-Arg-Phe (SEQ ID NO. 24). NPF polypeptidesof SEQ ID NOs. 5, 7, 9, 11, 13, and 15 are native NPF peptides frommosquito, American cockroach, or fruitfly, and exhibit homology with theamino acid sequences of SEQ ID NOs. 1 and 2, which are native to theColorado potato beetle. NPF polypeptides of SEQ ID NOs. 6, 8, 10, 12,14, and 16 are the respective non-amidated versions of these native NPFpeptides. Specifically exemplified NPF analogues are those comprising anamino acid sequence selected from the group consisting of SEQ ID NOs.17-24.

The term “pest” as used herein includes mosquitoes, insects and otherorganisms which adversely affect, humans, plants or animals, including,for example, organisms that remove blood, tissue and/or any other fluidfrom their prey or host. Pests controlled according to the subjectinvention specifically include those which regulate TTLE concentrationsin the gut by a mechanism which involves the binding of a ligand to areceptor to trigger an increase or a decrease in the synthesis ofdigestive enzymes, e.g., TMOF binding to its receptor. Examples of pestswhich can be controlled according to the subject invention include, butare not limited to, mosquitos, fleshflies, fleas, sandflies, houseflies,dogflies, and insects which attack plants.

The pest control compositions according to the subject inventioncomprise an NPF polypeptide, or a fragment, derivative, analogue orother functional equivalent of an NPF polypeptide, as a component, or asthe sole component. The pest control compositions may further comprise acarrier solution, compound, or molecule. Pest control compositions ofthe subject invention also include an NPF polypeptide, or a fragment,derivative, analogue or other functional equivalent of an NPFpolypeptide, contained in or associated with a cell, virus, plant, ormembrane. Examples include, but are not limited to, transformedbacteria, mammalian cells, algae, fungi, yeast viruses, or plants thatproduce an NPF polypeptide.

The term “functional equivalent” as used herein refers to a polypeptidesequence comprising full-length native NPF polypeptide, or a comprisingfragment, analogue (e.g., homologue), or derivative of a full-lengthnative NPF. Functional equivalents include, for example, an NPFpolypeptide in salt, complex, analogue, or derivative form as well as afragment, derivative or analogue of a native NPF peptide, which retainssome or all of the biological activity of the native NPF peptide.

The NPF polypeptides may be presented as fusion proteins or peptides,the amino acid sequence of which includes one or more NPF polypeptidesof the present invention. In various specific embodiments, two or moreof the NPF polypeptides are linked, for example, by peptide bondsbetween the N-terminus of one portion and the C-terminus of anotherportion. In other aspects, one or more of the NPF polypeptides can belinked to one or more heterologous polypeptides to form pesticidalfusion peptides. Molecules comprising such portions linked byhydrocarbon linkages are also provided. Derivatives of the foregoingfusion proteins are also provided (e.g., branched, curcularized,N-terminal carboxylated or C-terminal amidated).

In one embodiment the fusion protein or peptide comprises a repeatingunit of at least 4 amino acids (e.g., a multimer). There may be, forexample, from 2 to 10 or more repeating units. Preferably, the repeatingunit is connected through at least one amino acid which is cleaved by apest gut enzyme. Methods of recombinantly producing peptides in cells asmultimers are known in the art. (Rao et al., 1996 “Synthesis andexpression of genes encoding putative insect neuropeptide precursors intobacco,” Gene 175:1-5; Tortiglione et al., 1999 “New Genes for PestControl,” Genetics and Breeding for Crop Quality and Resistance,159-163). For example, a tandemly repeated DNA cassette for theexpression of NPF peptides can be constructed. As used herein, a pestgut enzyme is an enzyme which is present in the gut of a pest.Preferably, the pest is a mosquito or a lepidopteran. In a specificembodiment, the repeating units are connected through an arginine.

Analogues which have one or more amino acid substitutions forming abranched peptide (e.g., by substitution with an amino acid or amino acidanalogue having a free amino- or carboxy-side chain that forms a peptidebond with a sequence of one or more amino acids, including but notlimited to prolines) or allowing circularization of the peptide (e.g.,by substitution with a cysteine, or insertion of a cysteine at theamino- or carboxy-terminus or internally, to provide a sulfhydryl groupfor disulfide bond formation), are also provided.

Nonclassical amino acids or chemical amino acid analogues can replaceexisting amino acid residues of the NPF polypeptides or be inserted intothe NPF polypeptides between existing amino acid residues of the NPFpolypeptides or added to a terminus of the NPF polypeptides of thepresent invention. Non-classical amino acids include, but are notlimited to, the D-isomers of the common amino acids, 2,4-diaminobutyricacid, α-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyricacid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid,3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid,τ-butylglycine, τ-butylalanine, phenylglycine, cyclohexylalanine,β-alanine, fluoro-amino acids, designer amino acids such as β-methylamino acids, C-methyl amino acids, N-methyl amino acids, and amino acidanalogues in general. Furthermore, the amino acid can be D(dextrorotary) or L (levorotary). Dextrorotary amino acids are indicatedherein by a parenthetical D, i.e., “(D)”, immediately preceding thedextrorotary amino acid.

The NPF compounds include peptides containing, as a primary amino acidsequence, all or part of an exemplified NPF polypeptide sequence. TheNPF compounds thus include NPF polypeptides having conservativesubstitutions, i.e., altered sequences in which functionally equivalentamino acid residues are substituted for residues within the sequenceresulting in a peptide which is functionally active. For example, one ormore amino acid residues within the sequence can be substituted byanother amino acid of a similar polarity which acts as a functionalequivalent, resulting in a silent alteration. In one aspect of thepresent invention, conservative substitutions for an amino acid withinthe sequence may be selected from other members of the class to whichthe amino acid belongs (see Table 1). Conservative substitutions alsoinclude substitutions by amino acids having chemically modified sidechains which do not eliminate the pesticidal properties of the resultingNPF compound. TABLE 1 Class of Amino Acid Examples of Amino AcidsNonpolar Ala, Val, Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly,Ser, Thr, Cys, Tyr, Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

In a specific embodiment, the subject invention is directed toward amethod of controlling blood-ingesting pests comprising preparing atreatment comprising an NPF compound and applying said treatment to saidblood-ingesting pests. In another embodiment these peptides are used tocontrol agricultural pests.

Preparation of novel pest control compounds. The NPF polypeptides of theinvention can be prepared by well-known synthetic procedures. Forexample, the polypeptides can be prepared by the well-known Merrifieldsolid support method. See Merrifield (1963) J. Amer. Chem. Soc.85:2149-2154 and Merrifield (1965) Science 150:178-185. This procedure,using many of the same chemical reactions and blocking groups ofclassical peptide synthesis, provides a growing peptide chain anchoredby its carboxyl terminus to a solid support, usually cross-linkedpolystyrene or styrenedivinylbenzene copolymer. This method convenientlysimplifies the number of procedural manipulations since removal of theexcess reagents at each step is effected simply by washing of thepolymer.

Alternatively, these peptides can be prepared by use of well-knownmolecular biology procedures. Polynucleotides, such as DNA sequences,encoding the NPF polypeptides of the invention can be readilysynthesized. Such polynucleotides are a further aspect of the presentinvention. These polynucleotides can be used to genetically engineereukaryotic or prokaryotic cells, for example, bacteria cells, insectcells, algae cells, plant cells, mammalian cells, yeast cells or fungicells for synthesis of the peptides of the invention. Viruses may alsobe genetically modified using such polynucleotides, to serve as vectorsfor the delivery of the polynucleotides to insect pests or to othercells. One example of a cell line usefully transformed according to theteachings of the present invention is the insect cell line Sf9(Spodoptera frugiperda), deposit number ATCC CRL 1711, available fromthe American Type Culture Collection, 12301 Parklawn Drive, Rockville,Md. 20852 USA. An example of a useful virus includes the Baculovirus,Autographa californica Nuclear Polyhedrosis Virus (AcNPV), which isavailable from Texas A&M University, Texas Agricultural ExperimentStation, College Station, Tex. 77843, and described in Smithand Summers(1978) Virology 89:517-527; and (1979) J. Virology 30:828-838. Othernuclear polyhedrosis viruses (See World Health Organization TechnicalReport No. 531) such as Spodoptera frugiperda (Sf MNPV), Choristoneurafumiferana (Cf MNPV) (Smithand Summers [1981] J. Virol. 39:125-137), orSpodoptera littoralis (S1 NPV) (Harrap, et al. [1977] Virology 79:14-31)can be used instead of Autographa californica NPV. Other insect celllines can also be substituted for Spodoptera frugiperda (Sf9), forexample, Trichoplusia ni (Volkman and Summers [1975] J. Virol.16:1630-1637), Spodoptera exigua, Choristoneura fumiferana (Smith, andSummers [1981] J. Virol. 39:125-137) and Spodoptera littoralis (Harrap,K. A. et al. [1977] Virology 79:14-31).

In yet another embodiment, the subject invention is directed topolynucleotides which encode the subject NPF polypeptides.Polynucleotides can be produced by routine methods known in the art.[See S. L. Beaucage and M. H. Caruthers (1981), Tetrahedran Lett.22:1859].

The polynucleotides of the present invention can be amplified usingPolymerase Chain Reaction (PCR), a repetitive, enzymatic, primedsynthesis of a nucleic acid sequence. This procedure is well known andcommonly used by those skilled in this art (see Mullis, U.S. Pat. Nos.4,683,195, 4,683,202, and 4,800,159; Saiki, et al. [1985] “EnzymaticAmplification of β-Globin Genomic Sequences and Restriction SiteAnalysis for Diagnosis of Sickle Cell Anemia,” Science 230:1350-1354.).PCR employs the enzymatic amplification of a DNA fragment of interestthat is flanked by two oligonucleotide primers that hybridize toopposite strands of the target sequence. The primers are oriented withthe 3′ ends pointing towards each other. Repeated cycles of heatdenaturation of the template, annealing of the primers to theircomplementary sequences, and extension of the annealed primers with aDNA polymerase result in the amplification of the segment defined by the5′ ends of the PCR primers. Since the extension product of each primercan serve as a template another primer, each cycle essentially doublesthe amount of DNA fragment produced in the previous cycle, resulting inthe exponential accumulation of the specific target fragment, up toseveral million-fold in a few hours. By using a thermostable DNApolymerase such as Taq polymerase, which is isolated from thethermophilic bacterium Thermus aquaticus, the amplification process canbe completely automated. Other useful enzymes are known to those skilledin the art.

PCR primers can be designed from the DNA sequences of the subjectinvention. In performing PCR amplification, a certain degree of mismatchcan be tolerated between primer and template. Therefore, mutations,deletions, and insertions (especially additions of nucleotides to the 5′end) of the exemplified sequences fall within the scope of the subjectinvention. These PCR primers can be used to amplify genes of interestfrom a sampleproviding another method for identifying and characterizingpolynucleotide sequences encoding the NPF polypeptides.

The various methods employed in the preparation of the plasmids andtransformation of host organisms are well known in the art and aredescribed, for example, in U.S. Pat. Nos. 5,011,909 and 5,130,253. Thesepatents are incorporated herein by reference. These procedures are alsodescribed in Maniatis, et al. (1982) Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, New York. Thus, it is within theskill of those in the genetic engineering art to extract DNA from itssource, perform restrictions enzyme digestions, electrophorese DNAfragments, tail and anneal plasmid and insert DNA, ligate DNA, transformcells, e.g., E. coli or plant cells, prepare plasmid DNA, electrophoreseproteins, and sequence DNA.

Production of recombinant hosts. In another embodiment, the subjectinvention is directed to a cell transformed with a polynucleotideencoding a pesticidal NPF polypeptide. Hosts, which may be employedaccording to techniques well known in the art for the production of theNPF polypeptides of the present invention, include unicellularmicroorganisms, such as prokaryotes, i.e., bacteria; and eukaryotes,such as fungi, (including yeasts), algae, protozoa, molds, and the like,as well as plant cells, both in culture or in planta, and animal cells.Specific bacteria which are susceptible to transformation includemembers of the Enterobacteriaceae, such as strains of Escherichia coli;Salmonella; Bacillaceae, such as Bacillus subtilis; Pseudomonas;Pneumococcus; Streptococcus; Haemophilus influenzae, and yeasts such asSaccharomyces, among others.

In one embodiment of the present invention, the transformed host isBacillus sphaericus, which is known to be highly specific for control ofmosquito larvae. In a preferred embodiment, the host is Bacillussphaericus, serotype H5a5b, available from Abbott Laboratories asVectoLex CG Biological Larvicide (EPA Reg. No. 275-77).

The polynucleotide sequences of the subject invention can be introduceddirectly into the genome of the transformable host cell or can first beincorporated into a vector which is then introduced into the host.Exemplary methods of incorporation include transduction by recombinantphage or cosmids, transfection where specially treated host bacterialcells can be caused to take up naked phage chromosomes, andtransformation by calcium precipitation. These methods are well known inthe art. Exemplary vectors include plasmids, cosmids, and phages.

It is well known in the art that when synthesizing a gene for improvedexpression in a host cell it is desirable to design the gene such thatits frequency of codon usage approaches the frequency of preferred codonusage of the host cell. For purposes of the subject invention,“frequency of preferred codon usage” refers to the preference exhibitedby a specific host cell in usage of nucleotide codons to specify a givenamino acid. To determine the frequency of usage of a particular codon ina gene, the number of occurrences of that codon in the gene is dividedby the total number of occurrences of all codons specifying the sameamino acid in the gene. Similarly, the frequency of preferred codonusage exhibited by a host cell can be calculated by averaging frequencyof preferred codon usage in a large number of genes expressed by thehost cell. It is preferable that this analysis be limited to genes thatare highly expressed by the host cell.

Thus, in one embodiment of the subject invention, bacteria, algae,fungi, yeast, plants, or other cells can be genetically engineered,e.g., transformed with polynucleotides encoding the subject peptides toattain desired expression levels of the subject peptides. To providegenes having enhanced expression, the DNA sequence of the gene can bemodified to comprise codons preferred by highly expressed genes toattain an A+T content in nucleotide base composition which issubstantially that found in the transformed host cell. It is alsopreferable to form an initiation sequence optimal for the host cell, andto eliminate sequences that cause destabilization, inappropriatepolyadenylation, degradation and termination of RNA and to avoidsequences that constitute secondary structure hairpins and RNA splicesites. For example, in synthetic genes, the codons used to specify agiven amino acid can be selected with regard to the distributionfrequency of codon usage employed in highly expressed genes in the hostcell to specify that amino acid. As is appreciated by those skilled inthe art, the distribution frequency of codon usage utilized in thesynthetic gene is a determinant of the level of expression.

Assembly of the polynucleotide sequences of this invention can beperformed using standard technology known in the art. For example, astructural gene designed for enhanced expression in a host cell can beassembled within a DNA vector from chemically synthesizedoligonucleotide duplex segments. Preferably, the DNA vector or constructhas an operable promoter and suitable termination signals. Thepolynucleotide sequence can be introduced into a host cell and expressedby means known in the art. Preferably, the NPF compound produced uponexpression of the nucleotide sequence is functionally equivalent to thecorresponding purified NPF polypeptide. The present invention alsocomprises expression cassettes comprising the polynucleotides of thepresent invention and a expression vectors comprising polynucleotidesand/or expression cassettes of the present invention. According to thesubject invention, “functionally equivalent” indicates retention offunction such as, for example, pest control activity.

The present invention also includes chimeric polypeptides comprising oneor more heterologous polypeptides joined to one or more NPFpolypeptides, and also includes chimeric polypeptides comprising two ormore NPF polypeptides joined together. The portions which are combinedneed not, themselves, be pesticidal so long as the combination ofportions creates a chimeric protein which is pesticidal. The chimerictoxins may include portions from toxins which do not necessarily actupon the TMOF receptor including, for example, toxins from Bacillusthuringiensis (B.t.). B.t. toxins and their various toxin domains arewell known to those skilled in the art. Preferred toxins originate withvarious strains of B.t. including, for example, B.t. israeliensis, B.t.tenebrionis, B.t. san diego, B.t. aizawai, B.t. subtoxicus, B.t. alesti,B.t. gallaeriae, B.t. sotto, B.t. kurstaki, B.t. berliner, B.t.tolworthi, B.t. dendrolimus and B.t. thuringiensis, and other B.t.toxins known in the art such as the various delta-endotoxins describedin U.S. Pat. No. 5,686,069.

With the teachings provided herein, one skilled in the art can readilyproduce and use the various compounds and polynucleotide sequencesdescribed herein.

The polynucleotide sequences and compounds useful according to thesubject invention include not only the exemplified sequences but alsofragments of these sequences, variants, mutants, and fusion proteinswhich retain the characteristic pesticidal activity of the peptidesspecifically exemplified herein. As used herein, the terms “variants” or“variations” of genes refer to polynucleotides having differentnucleotide sequences but encoding the same polypeptides or encodingequivalent peptides having pesticidal activity. As used herein, the term“equivalent” in reference to a peptide or polypeptide refers tocompounds exhibiting some or all of the biological activity of nativeNPF peptides.

Variations of genes may be readily constructed using standard techniquesfor making point mutations. Also, fragments of these genes can be madeusing commercially available exonucleases or endonucleases according tostandard procedures. For example, enzymes such as the nuclease, BAL31,or site-directed mutagenesis can be used to systematically excisenucleotides from the ends of the genes. Also, genes which encode activefragments may be obtained using a variety of restriction enzymes.Proteases may be used to directly obtain active fragments of thesepeptides.

Polynucleotide sequences encoding NPF polypeptides can be introducedinto a wide variety of microbial or plant hosts with the result thatexpression of the gene results, directly or indirectly, in theproduction and maintenance of the NPF polypeptides. With suitablemicrobial hosts, e.g., yeast, Chlorella, the microbes can be applied tothe situs of the pest, where they will proliferate and be ingested bypest organisms, resulting in control of the pest. Alternatively, themicrobe hosting the gene can be killed and may optionally be treatedunder conditions that prolong the activity of the toxin and stabilizethe cell. Such killed cells can be applied to the habitat and/or to thehost or prey of the target pest. In one embodiment, the microbial orother host is transformed such that the gene encoding the pesticidal NPFpolypeptide is only exressed or maintained for a relatively short periodof time, such as days or weeks, so that the expression of the NPFpolypeptide does not continue indefinitely.

A wide variety of methods are available for introducing a polynucleotidesequence encoding a pesticidal polypeptide into a microorganism hostunder conditions which allow for stable maintenance and expression ofthe gene. These methods are well known to those skilled in the art andinclude, for example, the methods described in U.S. Pat. No. 5,135,867,which is incorporated herein by reference.

Synthetic genes which encode peptides which are functionally equivalentto the NPF polypeptide of the subject invention can also be used totransform hosts. Methods for the production of synthetic genes can befound in, for example, U.S. Pat. No. 5,380,831.

Recombinant cells expressing a pest control compound can be treated toprolong the pesticidal activity of the NPF polypeptide and stabilize thecell. For example, such cells can be formulated as a pesticidemicrocapsule comprising the NPF polypeptide within a stabilized cellularstructure that protects the toxin when the microcapsule is applied tothe environment of the target pest. Suitable host cells includeprokaryotes and eukaryotes. Preferred hosts include prokaryotes andlower eukaryotes, such as algae and fungi. The recombinant cell willpreferably be intact and be substantially in the proliferative form whentreated, rather than in a spore form.

Treatment of the microbial cell, e.g., a microbe containing thepolynucleotide sequence encoding the pesticidal polypeptide, can be bychemical or physical means, or by a combination of chemical and/orphysical means, so long as the technique does not completely diminishthe properties of the toxin, nor diminish the cellular capability ofprotecting the toxin. Methods for treatment of microbial cells aredisclosed in U.S. Pat. Nos. 4,695,455 and 4,695,462, which areincorporated herein by reference.

Methods and formulations for control of pests. Control of pests usingthe NPF polypeptides of the subject invention can be accomplished by avariety of methods known to those skilled in the art. These methodsinclude, for example, the application of recombinant microbes to thepests (or their locations), and the transformation of plants with genes(polynucleotide sequences) encoding the NPF polypeptides of the subjectinvention. Transformations can be made by those skilled in the art usingstandard techniques. Materials necessary for these transformations aredisclosed herein or are otherwise readily available to the skilledartisan.

The plant pests which can be controlled by the compounds of the subjectinvention generally belong to the phylum Arthropoda, including pests ofthe orders Coleoptera, Lepidoptera, Hemiptera and Thysanoptera. Otherpests which can be controlled according to the subject invention includemembers of the orders Diptera, Siphonaptera, Hymenoptera andPhthiraptera. Pests of the class Arachnida, such as ticks, mites, andspiders, can also be controlled by the NPF polypeptides of the presentinvention.

The use of the compounds of the subject invention to control pests canbe accomplished readily by those skilled in the art having the benefitof the instant disclosure. For example, the control compounds may beencapsulated, included in a granular form, solubilized in water or otherappropriate solvent, powdered, and included into any appropriateformulation for direct application to the pest. In a preferredembodiment for the control of plant pests, plants may be geneticallytransformed to express the pest control compound such that a pestfeeding upon the plant will ingest the control compound and thereby becontrolled.

Where the polynucleotide sequence is introduced via a suitable vectorinto a microbial host, and said host is applied to the environment in aliving state, it is preferred that certain host microbes be used.Microorganism hosts are selected which are known to occupy the“phytosphere” (phylloplane, phyllosphere, rhizosphere, and/orrhizoplane) of one or more crops of interest or the situs where the pestproliferates. These microorganisms are selected so as to be capable ofsuccessfully competing in the particular environment (crop and otherinsect habitats) with the wild-type organisms, provide for stablemaintenance and expression of the gene expressing the polypeptidepesticide, and, desirably, provide for improved protection of thepesticide from environmental degradation and inactivation.

A large number of microorganisms are known to inhabit the phylloplane(the surface of the plant leaves) and/or the rhizosphere (the soilsurrounding plant roots) of a wide variety of important crops. Thesemicroorganisms include bacteria, algae, and fungi. Preferredmicroorganisms include bacteria, e.g., genera Bacillus, Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes;fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium; andalgae, e.g., Chlorella. Of particular interest are such phytospherebacterial species as Pseudomonas syringae, Pseudomonas fluorescens,Serratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens,Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti,Alcaligenes entrophus, and Azotobacter vinlandii and Bacillusthurnigensis; and phytosphere yeast species such as Rhodotorula rubra,R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C.diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S.cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, andAureobasidium pollulans. Pigmented microorganisms are particularlypreferred.

Formulated bait granules containing an attractant and the NPFpolypeptides, or recombinant microbes comprising toxin-encodingpolynucleotide sequences, can be applied to the soil. Formulated productcan also be applied as a seed-coating or root treatment or total planttreatment at later stages of the crop cycle. Plant and soil treatmentsmay be employed as wettable powders, granules or dusts, by mixing withvarious inert materials, such as inorganic minerals (phyllosilicates,carbonates, sulfates, phosphates, and the like) or botanical materials(powdered corncobs, rice hulls, walnut shells, and the like). Theformulations may include spreader-sticker adjuvants, stabilizing agents,other pesticidal additives, or surfactants. Liquid formulations may beaqueous-based or non-aqueous and employed as foams, gels, suspensions,emulsifiable concentrates, or the like. The ingredients may includerheological agents, surfactants, emulsifiers, dispersants, or polymers.

As would be appreciated by a person skilled in the art, theconcentration of NPF polypeptide in the pesticidal formulations of thepresent invention will vary widely depending upon the nature of theparticular formulation, particularly whether it is a concentrate or tobe used directly, and on the potency of the NPF polypeptide(s) selected.The NPF polypeptide will be present in at least about 0.0001% by weightand may be 100% by weight. The dry formulations will have from about0.0001-95% by weight of the NPF polypeptide, while the liquidformulations will generally be from about 0.0001-60% by weight of thesolids in the liquid phase. The formulations that contain cells willgenerally have from about 1 to about 10⁴ cells/mg. These formulationswill be administered at about 50 mg (liquid or dry) to 1 kg or more perhectare.

The formulations can be applied to the environment of the pest, e.g.,soil and foliage, by spraying, dusting, sprinkling, or the like, and/orto hosts or prey of the pests, e.g., plants, and humans or otheranimals.

In applications to the environment of the target pest, a transformantstrain can be applied to the natural habitat of the pest. In some cases,the transformant strain will continue to grow in the pest upon ingestionand produce NPF polypeptide following ingestion by the pest. Thetransformed organism may be applied by spraying, soaking, injection intothe soil, seed coating, seedling coating or spraying, or the like. Whereadministered in the environment, concentrations of the organism willgenerally be from 1 to 10¹⁰ cells/ml, and the volume applied per hectarewill be generally from about 0.1 oz to 2 lbs or more. Where administeredto a plant part inhabited by the target pest, the concentration of theorganism will usually be from 10³ to 10⁶ cells/cm².

In aquatic environments, pest control may be attained at or below thesurface by adjusting the specific gravity of the microbe. This can beaccomplished by, for example, varying the lipid content of thetransformant microorganism strain. It is known that indigenous aquaticalgae float due to their lipid content. A variation in lipid contentwill allow the transformant strain to be distributed at desired depthsbelow the water surface.

For commercial formulations, the organisms may be maintained in anutrient medium which maintains selectivity and results in a low rate ofproliferation. Various media may be used, such as yeast extract orL-broth. Once the organism is to be used in the field, thenon-proliferating concentrate may be introduced into an appropriateselective nutrient medium, grown to high concentration, generally fromabout 10⁵ to 10⁹ cells/ml and may then be employed for introduction intothe environment of the pest.

All of the U.S. patents and other references cited herein are herebyincorporated by reference, as are U.S. patent application Ser. No.09/295,846, (UF-223) “Transformed Cells Useful for the Control ofPests”; U.S. patent application Ser. No. 09/551,737, (UF-223C1)“Transformed Cells Useful for the Control of Pests”; U.S. patentapplication Ser. No. 09/296,113, (UF-224) “Materials and Methods Usefulfor the Control of Insect Larvae”; U.S. patent application Ser. No.09/551,738, (UF-224C1) “Materials and Methods Useful for the Control ofInsect Larvae”; U.S. patent application Ser. No. 09/295,996, (UF-230)“Novel Peptides and the Use Thereof to Control Pests”; and U.S. patentapplication Ser. No. 09/295,924, (IPTL Docket No. 4137-120)“Compositions and Methods for Controlling Pests”.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Effect of NPF Polypeptides on Trypsin Biosynthesis

To find out if NPF I and II affect trypsin biosynthesis in the midgut offemale Aedes aegypti, females were fed a blood meal and immediatelyinjected with 0.25 μl of the peptide at concentrations of 2.5 μg to 12.5μg and 30 hours later the midguts were removed and assayed for trypsinbiosynthesis (Borovsky et al., 1990 “Mosquito Oostatic Factor: A NovelDecapeptide Modulating Trypsin-Like Enzyme Biosynthesis in the Midgut”FASEB J. 4:3015-3020; Borovsky et al. 1993 “Mass Spectrometry andCharacterization of Aedes aegypti Trypsin Modulating Oostatic Factor(TMOF) and its Analog” Insect Biochem. Molec. Biol. 23:703-712). Eachexperiment was repeated 3 times (5 females per group) and the resultsare expressed as % inhibition of trypsin biosynthesis±S.E.M. (FIG. 1).Fifty percent inhibition of trypsin biosynthesis was achieved at aconcentration of 10⁻⁶M NPF I. NPF II was effective at a dose of 10⁻³M(78%±10), at 10⁻⁶M NPF II inhibited trypsin biosynthesis by 35% (FIG.2).

To determine if NPF I releases a neuroendocrine factor from the brain orthe thoracic ganglia which in turn may release TMOF from the ovary,female Aedes aegypti were fed a blood meal, immediately ligated andinjected with different concentrations of NPF I(10⁻³M to 10⁻⁹M) in 0.2582 l of sterile distilled water. Thirty hours later, abdomens wereremoved and 3 groups of 5 abdomens per NPF concentration were assayedfor trypsin biosynthesis (Borovsky et al., 1990, 1993). Fifty-fourpercent inhibition was achieved with 10⁻⁶M of NPF I indicating that NPFI affects trypsin biosynthesis in the gut by binding to a TMOF receptorand not by the release of neuroendocrine factors from the brain or thethoracic ganglia that in turn release TMOF from the ovary. Because thestructure of NPF I is different from TMOF it appears that NPF I does notbind to TMOF specific binding site on the gut receptor but to adifferent site on the same or different receptor.

EXAMPLE 2 Effect of NPF Polypeptides on Mosquito Larvae

NPF polypeptides of the subject invention have been found to be highlyeffective pest control agents. NPF polypeptides sharing sequencehomology with NPF I and NPF II of the Colorado potato beetle(Leptinotarsa decemlineata) (Spittaels, et al. Insect Biochem. Mol.Biol., 26(4):375-382, 1996) have been identified in the Americancockroach (Periplaneta americana) (Veenstra and Lambrou, Biochem.Biophys. Res. Commun., 213(2):519-524, 1995), mosquito (Aedes aegypti)(Stanek et al., Display Presentation, Entomological Society of AmericaAnnual Meeting, Montreal, Canada, Dec. 3-6, 2000), and fruit fly(Drosophila melanogaster), and their pesticidal activity was confirmed.Synthesized polypeptides corresponding to the native polypeptides ofColorado potato beetle (SEQ ID NO. 1), mosquito (SEQ ID NOs. 5 and 7),American cockroach (SEQ ID NOs. 9 and 11), and fruit fly (SEQ ID NOs. 13and 15) were provided to first instar Aedes aegypti larvae foringestion. Non-amidated versions of SEQ ID NOs. 5, 7, 9, 11, 13, and 15(SEQ ID NOs. 6, 8, 10, 12, 14, and 16, respectively), as well as otherfunctional equivalents (SEQ ID NOs. 17-24), were also provided foringestion.

First instar Aedes aegypti larvae were assayed in a microtiter plate in188 μl solution containing 160 μl of water, 10 μl of 2% Brewer's yeast,and NPF polypeptides of the subject invention at concentrations of 2mg/ml to 0.04 mg/ml (Table 2). Mortality was determined at 24 hourintervals for 3 to 7 days. Controls were run with yeast solution lackingthe NPF polypeptide. TABLE 2 Effect of NPF polypeptides on Aedes aegyptilarvae LC₅₀ (mM) SEQ ID NO. NPF Polypeptide ± SEM SEQ ID NO. 1Ala—Arg—Gly—Pro—Gln—Leu—Arg—Leu—Arg—Phe—NH₂  0.65 ± 0.040 (Mol. Wt.:1211.30) SEQ ID NO. 5 Arg—Pro—Pro—The—Arg—Phe—Arg—Phe—NH₂  0.61 ± 0.018(Mol. Wt.: 1074.23) SEQ ID NO. 6 Arg—Pro—Pro—Thr—Arg—Phe—Arg—Phe—OH 0.26 ± 10.02 (Mol. Wt.: 1076.22) SEQ ID NO. 7Ala—Pro—Gln—Leu—Arg—Leu—Arg—Phe—NH₂  0.8 ± 0.06 (Mol. Wt.: 999.2) SEQ IDNO. 8 Ala—Pro—Gln—Leu—Arg—Leu—Arg—Phe—OH 0.65 ± 0.06 (Mol. Wt.: 1000.18)SEQ ID NO. 9 Ala—Asn—Arg—Ser—Pro—Ser—Leu—Arg—Leu—Arg—Phe—NH₂ 0.135± 0.06  (Mol. Wt.: 1315.49) SEQ ID NO. 10Ala—Asn—Arg—Ser—Pro—Ser—Leu—Arg—Leu—Arg—Phe—OH  0.68 ± 0.037 (Mol. Wt.:1316.47) SEQ ID NO. 11 Ala—Asp—Arg—Ser—Pro—Ser—Leu—Arg—Leu—Arg—Phe—NH₂0.65 ± 0.03 (Mol. Wt.: 1315.47) SEQ ID NO. 12Ala—Asp—Arg—Ser—Pro—Ser—Leu—Arg—Leu—Arg—Phe—OH 0.607 ± 0.066 (Mol. Wt.:1317.45) SEQ ID NO. 13 Pro—Ile—Arg—Ser—Pro—Ser—Leu—Arg—Leu—Arg—Phe—NH₂0.589 ± 0.04  (Mol. Wt.: 1253.52) SEQ ID NO. 14Pro—Ile—Arg—Ser—Pro—Ser—Leu—Arg—Leu—Arg—Phe—OH 0.558 ± 0.03  (Mol. Wt.:1254.5) SEQ ID NO. 15 Ala—Gln—Arg—Ser—Pro—Ser—Leu—Arg—Leu—Arg—Phe—NH₂1.03 ± 0.1  (Mol. Wt.: 1329.52) SEQ ID NO. 16Ala—Gln—Arg—Ser—Pro—Ser—Leu—Arg—Leu—Arg—Phe—OH 1.187 ± 0.11  (Mol. Wt.:1330.5) SEQ ID NO. 17 Arg—Ser—Pro—Ser—Leu—Arg—Leu—Arg—Phe—NH₂ 0.82± 0.07 (Mol. Wt.: 1130.32) SEQ ID NO. 18Arg—Ser—Pro—Ser—Leu—Arg—Leu—Arg—Phe—OH 0.76 ± 0.04 (Mol. Wt.: 1131.3)SEQ ID NO. 19 Pro—Ser—Leu—Arg—Leu—Arg—Phe—NH₂ >2.25 (Mol. Wt.: 887.07)SEQ ID NO. 20 Pro—Ser—Leu—Arg—Leu—Arg—Phe—OH  0.76 ± 0.051 (Mol. Wt.:888.05) SEQ ID NO. 21 Leu—Arg—Leu—Arg—Phe—NH₂ >2.8  (Mol. Wt.: 702.89)SEQ ID NO. 22 Leu—Arg—Leu—Arg—Phe—OH 1.589 ± 0.143 (Mol. Wt.: 703.87)SEQ ID NO. 23 Arg—Pro—Pro—Thr—OH 2.08 ± 0.15 (Mol. Wt.: 469.52) SEQ IDNO. 24 Arg—Phe—Arg—Phe—OH 0.81 ± 0.06 (Mol. Wt.: 624.72)

EXAMPLE 3 Cytoimmunochemical Analysis

Cytoimmunochemical analysis of the mosquito gut after the blood mealusing antiserum against NPF I revealed that exocrine cells with NPFI-like molecules are synthesized by the mosquito epithelial cells 24hours after a blood meal. In females that did not take a blood mealthese cells are not found. Thus, it is possible that NPF I is asecondary signal in a cascade of signals that starts with the release ofTMOF from the ovary, the hormone then binds to a TMOF gut receptor(Borovsky et al., 1994) that stimulates the synthesis and release of NPFI from gut specific exocrine cells. NPF I binds to a receptor NPF Ibinds to a receptor site on the gut the binding site may be adjacent toor part of the TMOF receptor and causes the cessation of trypsinbiosynthesis.

EXAMPLE 4 Bioassays for Activity Against Lepidopteron and Coleopterans

Biological activity of the pest control compounds of the subjectinvention can be confirmed using standard bioassay procedures. One suchassay is the budworm-bollworm (Heliothis virescens [Fabricius] andHelicoverpa zea [Boddie]) assay. Lepidoptera bioassays can be conductedwith either surface application to artificial insect diet or dietincorporation of samples. All Lepidopteran insects can be tested fromthe neonate stage to the second instar. All assays can be conducted witheither toasted soy flour artificial diet or black cutworm artificialdiet (BioServ, Frenchtown, N.J.).

Diet incorporation can be conducted by mixing the samples containing thepest-control compound with artificial diet at a rate of 6 mL suspensionplus 54 mL diet. After vortexing, this mixture is poured into plastictrays with compartmentalized 3-ml wells (Nutrend Container Corporation,Jacksonville, Fla.). A water blank containing no pest control compoundserves as the control. First instar larvae (USDA-ARS, Stoneville, Miss.)are placed onto the diet mixture. Wells are then sealed with Mylarsheeting (ClearLam Packaging, Ill.) using a tacking iron, and severalpinholes are made in each well to provide gas exchange. Larvae can beheld at 25° C. for 6 days in a 14:10 (light:dark) holding room.Mortality and stunting are then recorded after six days.

Bioassay by the top load method utilizes the same sample and dietpreparations as listed above. The samples are applied to the surface ofthe insect diet. In a specific embodiment, surface area can range from0.3 to approximately 0.8 cm² depending on the tray size; 96 well tissueculture plates can be used in addition to the format listed above.Following application, samples are allowed to air dry before insectinfestation. A water blank containing no control compound can serve asthe control. Eggs are applied to each treated well. The wells are thensealed with Mylar sheeting (ClearLam Packaging, Ill.) using a tackingiron, and pinholes are made in each well to provide gas exchange.Bioassays are held at 25° C. for 7 days in a 14:10 (light:dark) or 28°C. for 4 days in a 14:10 (light:dark) holding room. Mortality and insectstunting are recorded at the end of each bioassay.

Another assay useful according to the subject invention is the Westerncorn rootworm assay. Samples can be bioassayed against neonate westerncorn rootworm larvae (Diabrotica virgifera virgifera) via top-loading ofthe pest control sample onto an agar-based artificial diet at a rate of160 ml/cm². Artificial diet can be dispensed into 0.78 cm² wells in48-well tissue culture or similar plates and allowed to harden. Afterthe diet solidifies, samples are dispensed by pipette onto the dietsurface. Excess liquid is then evaporated from the surface prior totransferring approximately three neonate larvae per well onto the dietsurface by camel's hair brush. To prevent insect escape while allowinggas exchange, wells are heat-sealed with 2-mil punched polyester filmwith 27HT adhesive (Oliver Products Company, Grand Rapids, Mich.).Bioassays are held in darkness at 25° C., and mortality scored afterfour days.

Analogous bioassays can be performed by those skilled in the art toassess activity against other pests, such as the black cutworm (Agrotisipsilon).

EXAMPLE 5 Target Pests

Toxins of the subject invention can be used, alone or in combinationwith other toxins, to control one or more non-mammalian pests. Thesepests may be, for example, those listed in Table 3. Activity can readilybe confirmed using the bioassays provided herein, adaptations of thesebioassays, and/or other bioassays well known to those skilled in theart. TABLE 3 Target pest species ORDER/Common Name Latin NameLEPIDOPTERA European Corn Borer Ostrinia nubilalis European Corn Borerresistant to Cry1A Ostrinia nubilalis Black Cutworm Agrotis ipsilon FallArmyworm Spodoptera frugiperda Southwestern Corn Borer Diatraeagrandiosella Corn Earworm/Bollworm Helicoverpa zea Tobacco BudwormHeliothis virescens Tobacco Budworm Rs Heliothis virescens SunflowerHead Moth Homeosoma ellectellum Banded Sunflower Moth Cochylis hospesArgentine Looper Rachiplusia nu Spilosoma Spilosoma virginica BerthaArmyworm Mamestra configurata Diamondback Moth Plutella xylostellsCOLEOPTERA Red Sunflower Seed Weevil Smicronyx fulvus Sunflower StemWeevil Cylindrocopturus adspersus Sunflower Beetle Zygorammaexclamationis Canola Flea Beetle Phyllotreta cruciferae Western CornRootworm Diabrotica virgifera virgifera DIPTERA Hessian Fly Mayetioladestructor HOMOPTERA Greenbug Schizaphis graminum HEMIPTERA Lygus BugLygus lineolaris NEMATODA Heterodera glycines

EXAMPLE 6 Insertion of Toxin Genes Into Plants

One aspect of the subject invention is the transformation of plants withgenes encoding the insecticidal toxin of the present invention. Thetransformed plants are resistant to attack by the target pest.

Genes encoding pesticidal toxins, as disclosed herein, can be insertedinto plant cells using a variety of techniques which are well known inthe art. For example, a large number of cloning vectors comprising areplication system in E. coli and a marker that permits selection of thetransformed cells are available for preparation for the insertion offoreign genes into higher plants. The vectors comprise, for example,pBR322, pUC series, M13mp series, pACYC184, etc. Accordingly, thesequence encoding the Bacillus toxin can be inserted into the vector ata suitable restriction site. The resulting plasmid is used to transformE. coli. The E. coli cells are cultivated in a suitable nutrient medium,then harvested and lysed. The plasmid is recovered. Sequence analysis,restriction analysis, electrophoresis, and other biochemical and/ormolecular biological methods are generally carried out as methods ofanalysis. After each manipulation, the DNA sequence used can be cleavedand joined to the next DNA sequence. Each plasmid sequence can be clonedin the same or other plasmids. Once the inserted DNA has been integratedin the genome, it is relatively stable there and, as a rule, does notcome out again. It normally contains a selection marker that confers onthe transformed plant cells resistance to a biocide or an antibiotic,such as kanamycin, G418, bleomycin, hygromycin, or chloramphenicol,inter alia. The individually employed marker should accordingly permitthe selection of transformed cells rather than cells that do not containthe inserted DNA.

A large number of techniques are available for inserting DNA into aplant host cell. These techniques include transformation with T-DNA(“transferred DNA”; discussed in more detail below) using Agrobacteriumtumefaciens or Agrobacterium rhizogenes as transformation agent, fusion,injection, biolistics (microparticle bombardment), or electroporationand other methods known to those of skill in the art.

One of the most widely used approaches for the introduction of DNA intoplant cells exploits the natural DNA-transferring properties ofAgrobacterium tumefacients and Agrobacterium rhizogenes, the two specieswhich cause crown gall and hairy root. Their ability to cause diseasedepends on the presence of large plasmids, in excess of 100 kb, whichare referred to as the “Tumour-inducing” or (Ti) and “Root-inducing” (orRi) plasmids respectively.

A region referred to as the T-DNA (“Transferred DNA”) is transferredfrom an infecting Agrobacterium cell into the nucleus of the plant cell,where it is integrated into the plant genome. Transfer of the T-DNAdepends on a set of genes called vir if they are on the Ti plasmid, orchv if they are on the chromosome. These genes are induced in responseto various compounds in exudates from wounded plants. The T-DNA itselfis flanked by repeated sequences of around 25 base pairs, called borderrepeats (or left and right borders). The T-DNA contains a group of genesreferred to as the onc genes, which are responsible for the oncogenicityof the T-DNA.

The use of Agrobacterium in the genetic manipulation of plants involvesthe insertion of foreign DNA into the T-DNA of a bacterial cell andsubsequent transfer of the DNA by the transformed bacterium into theplant. As long as the necessary proteins are provided by the bacterium,any sequences flanked by the T-DNA border repeats can be transferredinto the recipient plant cell genome. The Ti plasmids are too large tomanipulate directly, but this problem can be circumvented by usingcointegrative and binary systems.

The two main components of a cointegrative system are a Ti plasmid thathas typically been modified by the replacement of material between theborder repeats (including the onc sequences) by pBR322; and aintermediate vector, which is a modified pBR322 containing an extramarker, such as kanamycin resistance. The gene to be introduced into thetarget plant is first cloned in to the intermediate vector, and thisconstruct is then introduced into Agrobacterium containing the Tivector. The pBR322-based plasmid cannot replicate efficiently insideAgrobacterium, so selection for kanamycin resistance identifies thoseAgrobacterium cells where the pBR322-based intermediate plasmid has beenintegrated by homologous recombination into the Ti plasmid. Because therecombination is homologous, it will take place across the pBR322sequences and therefore result in integration between the borderrepeats.

The need for cointegration of the plasmids can be circumvented by use ofa binary vector, such as pBin19, a small plasmid containing a pair ofleft and right borders. The lacZ region, located within the borders,facilitates insertion and detection of DNA. A neomycinphosphotransferase gene, typically modified for expression in plants byaddition of nopalline synthase expression sequences, is also presentwithin the borders. Outside the left and right borders, there istypically a kanamycin resistance gene that will function in prokaryotesand a broad host-range origin derived from the plasmid pRK252. Theproteins that catalyze transfer of the T-DNA into the host plant do nothave to be cis-encoded (i.e., do not have to be encoded by the samemolecule). Therefore, if the binary vector is introduced intoAgrobacterium that already contains a resident Ti plasmid, the residentplasmid can provide all the functions needed to transfer into a plantnucleus the DNA between the borders of the binary vector.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

1. An isolated polypeptide comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQID NO:24.
 2. The polypeptide of claim 1, wherein said polypeptideconsists of an amino acid sequence selected from the group consisting ofSEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ IDNO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24.
 3. The polypeptideof claim 1, wherein said polypeptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQID NO:24.
 4. The polypeptide of claim 1, wherein said polypeptideconsists of an amino acid sequence selected from the group consisting ofSEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24.
 5. The polypeptide ofclaim 1, wherein said polypeptide is a fusion polypeptide comprising anNPF polypeptide and a heterologous polypeptide, and wherein said NPFpolypeptide comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24.
 6. Apesticidal composition comprising a pesticidal polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, and SEQ ID NO:24; and a pesticidally effectivecarrier.
 7. The pesticidal composition of claim 6, wherein saidpesticidal polypeptide consists of an an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQID NO:24.
 8. The pesticidal composition of claim 6, wherein saidpolypeptide is a fusion polypeptide comprising a pesticidal polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24; and a heterologouspolypeptide.
 9. The pesticidal composition of claim 6, wherein saidcomposition further comprises a pest attractant.
 10. The pesticidalcomposition of claim 6, wherein said composition further comprisesbotanical material.
 11. The pesticidal composition of claim 6, whereinsaid composition further comprises an ingredient selected from the groupconsisting of a spreader-sticker adjuvant, stabilizing agent, andsurfactant.
 12. The pesticidal composition of claim 6, wherein saidcomposition further comprises a pesticidal additive.
 13. The pesticidalcomposition of claim 6, wherein said composition further comprises aningredient selected from the group consisting of a rheological agent,emulsifier, dispersant, and polymer.
 14. The pesticidal composition ofclaim 6, wherein said composition is a foam, gel, suspension, oremulsifiable concentrate.
 15. A method of controlling a pest, comprisingapplying to the pest, or to a pest-inhabited locus, a pesticidallyeffective amount of an NPF polypeptide.
 16. The method of claim 15,wherein the NPF polypeptide comprises an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQID NO:24.
 17. The method of claim 15, wherein the NPF polypeptideconsists of an amino acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24.
 18. The method of claim15, wherein the method comprises applying to the pest, or to apest-inhabited locus, a fusion polypeptide comprising the NPFpolypeptide and a heterologous polypeptide.
 19. The method of claim 15,wherein the NPF polypeptide comprises a dextrorotary amino acid.
 20. Themethod of claim 15, wherein the pest is selected from the groupconsisting of coleopterans, lepidopterans, and dipterans.
 21. The methodof claim 15, wherein the pest is a blood-sucking pest.
 22. The method ofclaim 15, wherein the pest is a pest of the suborder Nematocera.
 23. Themethod of claim 15, wherein the pest is a pest of the family Colicidae.24. The method of claim 15, wherein the pest is a dipteran.
 25. Themethod of claim 15, wherein the pest is a pest of a genus selected fromthe group consisting of Heliothis, Culex, Theobaldia, Aedes, Anopheles,Forciponiyia, Culicoides and Helea.
 26. The method of claim 15, whereinthe pest is selected from the group consisting of mosquitoes, fleshflies, fleas, sand flies, house flies, and dog flies.
 27. The method ofclaim 15, wherein the pest is a mosquito.
 28. The method of claim 15,wherein the pest is a pest species selected from the group consistingof: Aedes aegypti, Culex quinquefasciatus, Anopheles albimanus,Anopheles quadrimaculatus, Lutzomyia anthrophora, Culicoidesvariipennis, Stomoxys calcitrans, Musca domestica, Ctenocephalidesfelis, and Heliothis virescens.
 29. The method of claim 15, wherein themethod comprises applying the NPF polypeptide to a pest-inhabited locus,and wherein the pest-inhabited locus is a body of water.
 30. The methodof claim 15, wherein the pest utilizes a serine esterase as a digestiveenzyme, wherein the pest ingests the NPF polypeptide, and wherein theNPF polypeptide inhibits synthesis of the serine esterase within thepest.
 31. The method of claim 15, wherein the pest utilizes trypsin as adigestive enzyme, wherein the pest ingests the NPF polypeptide, andwherein the NPF polypeptide inhibits synthesis of the trypsin within thepest.